| Autor: |
Ruby Sukhraj, Josh E. Baker, Christine P. Cremo, Michael S. Carter, Feng Hong, Michael P. Walsh, Mariam Ba |
| Rok vydání: |
2012 |
| Předmět: |
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| Zdroj: |
Biophysical Journal. 102(3):358a-359a |
| ISSN: |
0006-3495 |
| DOI: |
10.1016/j.bpj.2011.11.1959 |
| Popis: |
We are interested in the mechanism of phosphorylation of smooth muscle myosin (SMM) by the myosin light chain kinase -calmodulin-Ca2+ complex (MLCK-CaM-Ca2+). This reaction is required for activation of SMM catalytic activity and smooth muscle contraction. In previous studies we characterized tightly-bound SMM-MLCK-CaM complexes in an in vitro model system and demonstrated that SMM-MLCK-CaM complexes co-purified from smooth muscle were functional, i.e. MLCK was able to phosphorylate SMM and the phosphorylated SMM resulted in actin filament motility in an in vitro assay. Moreover, using total internal reflectance fluorescence microscopy (TIRF), we observed dynamic interactions between single MLCK and SMM molecules by visualizing quantum dot-labeled MLCK (QD-MLCK) interacting with SMM aligned on actin filaments.We have also observed that QDs-MLCK moved along the pure actin filaments. We are currently using cultured human airway smooth muscle cells, which is a more physiological system, to determine the mechanism whereby one MLCK phosphorylates many SMM molecules. Direct observations of single QD-labeled MLCK molecules show clearly that MLCK co-localizes with and can move along the actin- and myosin-containing stress fibers, under the conditions of high ionic strength, or physiological ionic strength with CaM-Ca2+ and ATP. The diffusion coefficient, calculated from mean-squared-displacement (MSD) data from MLCK-QDs’ trajectories, indicates that the mechanism by which one MLCK phosphorylates multiple SMMs may involve MLCK movement along thick and/or thin filaments on a time scale measured in seconds. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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